Sample preparation is a critical step in the analysis of complex matrices for trace components, particularly in the area of life sciences. Solid phase extraction (SPE) techniques can be valuable to an analyst solving problems relating to sample concentration, sample clean-up and analyte isolation. SPE is recognized as a desirable alternative to liquid-liquid extraction (LLE) because SPE minimizes or eliminates altogether the use of organic solvents, which are regulated as priority pollutants. Further, LLE can lead to emulsion formation and if particulates are present in a sample, adsorption of analyte onto these structures can result in low recoveries. Compared with LLE, SPE can offer a more complete extraction of analytes, a more efficient separation of interferences from analytes, easier collection of total analyte fraction and removal of particulates and can be more easily automated. Solid phase extraction is presently extensively applied in separations performed in widely differing fields, including, but not limited to environmental pollution, agrochemicals, discovery and/or development of pharmaceuticals, analytical toxicology, the development of nutritional products, drinking water purity assessment and biotechnology. Several individual monographs, journal review articles and research publications on the theory and practice of SPE technology have been published (see, e.g., Thurman & Mills, (1998) Solid Phase Extraction, Wiley, New York, N.Y.; Simpson (Ed.), (2000) Solid Phase Extraction, Marcel Dekker, New York, N.Y.; J Chromatog. A. (2000) 885: entire issue; Snyder, Kirkland & Glajch, Practical HPLC Method Development, Chapter 4, pp 100-173, Wiley, New York, N.Y., 1997).
Solid phase extraction protocols followed by academic, industrial and government laboratories typically employ syringe-barrel cartridges, which can include cartridges designed for syringe use, as well as disks and disk cartridges (see, e.g., Thurman & Snavely, (2000) Trend Anal. Chem. 19:18-26), thin packed bed syringe-barrel cartridges, solid phase microextraction fibers (for both gas chromatographic (GC) and high performance liquid chromatography (HPLC) applications), 96-well plates, SPE pipette tips, and robot-compatible large reservoirs. The syringe barrel device format is the most commonly employed format, followed by the disk format. The disk format facilitates the use of higher flow rates, due to their large cross-sectional areas and shorter bed depths, and utilize very small elution solvent volumes. For drug screening and clinical trial applications, both of which require high sample throughput and utilize liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) as the primary analytical tool, the multi-well plate format (e.g. 96-well plates, 384-well plates and 1536-well plates) has gained popularity.
Silica and related bonded phases constituted the dominant SPE sorbents until about 1996, as evidenced by the extensive application bibliographies prepared by several SPE material manufacturers (e.g., Varian Sample Preparation Products, Harbor City, Calif., 1995; Bakerbond SPE Bibliography, JTBaker, Inc, Philipsburg, N.J., 1995; McDonald & Bouvier, (Eds.), Solid Phase Extraction Applications Guide and Bibliography: A Resource for Sample Preparation Methods Development, Waters Corp., Milford, Mass. 6th ed., 1995). During the last few years, however, many polymeric sorbents have been introduced for sample pretreatment applications (see, e.g., U.S Pat. No. 5,618,438; U.S. Pat. No. 5,882,521; and U.S. Pat. No. 6,106,721; Fritz & Macka, (2000) J. Chromatog. A 902:137-166). Some of these polymeric sorbents are based on a styrene divinylbenzene or methacrylate polymeric backbone. Advantages of polymeric SPE sorbents over their silica-based counterparts include their stability to pH extremes and their higher surface area, which can facilitate greater capacity and retention than observed for silica-based materials. In addition, silica-based materials comprise silanol groups. These groups can complicate analyte retention, due to the influence of the pH and ionic strength of the sample matrix on the silanol groups.
One limitation of commercially available reversed-phase silica sorbents, as well as the first generation of styrene-divinylbenzene polymers, is the need for conditioning them with a wetting solvent and the additional requirement that they remain wetted prior to sample loading. The advent of second generation polymeric sorbents comprising polar functional groups such as sulfonic/carboxylic acid, hydroxymethyl, keto, nitro and heterocyclic amide moieties ameliorates these requirements due to the capacity of these polar groups to adsorb and retain water on their surface.
These reversed-phase silica and second generation polymeric materials are not, however, without problems. A major shortcoming of reversed-phase silicas and second generation polymers is the inability of these materials to retain polar compounds, such as some drug metabolites and pharmaceuticals. Many of these SPE materials exhibit unacceptable breakthrough for polar molecules during the loading and/or washing steps, resulting in poor analyte recoveries. This phenomenon places severe limitations on the applicability of SPE protocols for analyte extraction and sample clean-up when the sample comprises a mixture of an analyte, which can be hydrophobic, and its metabolites or degradation products, which tend to be very polar. Moreover, the pharmaceutical industry is designing more products with significant polar characteristics. The inadequate retention of such drugs on a polymeric sorbent during sample pretreatment can lead to serious problems.
Another limitation of prior art polymeric sorbents is in the area of ion suppression. Several publications highlight an ion suppression effect observed during LC/MS/MS analysis of drugs in biological matrices (see, e.g., Bonfiglio et al., (1999) Rapid Commun. Mass Sp. 13: 1175-1185; King et al., (2000) J Am. Soc. Mass Spectr. 11: 942-950). These publications attribute the observed ion suppression to the presence of matrix constituents left behind on an SPE sorbent during sample loading and washing steps. These constituents can then contaminate desired extracts during analyte elution. During LC/MS, polar drugs elute from the LC column either with these matrix constituents or closely after elution of the matrix constituents. These polar drugs can be severely affected by ion suppression, rendering their quantitation unreliable. Thus, another problem associated with prior art sorbents is the presence of unacceptable levels of ion suppression.
Yet another problem associated with prior art SPE materials is the limitation on the amount of organic component that can be employed to wash (or elute) an analyte of interest after a sample comprising the analyte is applied to a prior art polymeric sorbent. Procedures for employing prior art SPE materials typically recommend the use of aqueous solvents and buffers containing a low percentage of an organic component (<5%) for washing the SPE material after a sample has been loaded onto the material. These procedures recommend a low percentage of organic component because if the organic content is increased too much, this can lead to the almost complete removal of the more polar constituents of the sample, including an analyte of interest. This is due, in part, to the inability of prior art sorbents to retain moderately to highly polar compounds. A few commercial polymeric sorbents, such as those comprising sulfonic acid moieties, are known to enhance polar retention through ionic mechanisms. SPE protocols using these sorbents are tedious, however, and such elutions are typically carried out with solvents that are incompatible with mass spectrometric detectors.
Thus, there is a need for a polymeric sorbent that strongly retains moderately to highly polar analytes, particularly when the analytes are present in a complex matrix (e.g. a biological, environmental or pharmaceutical sample). There is also a need for a polymeric sorbent that can be treated with solvents comprising a high percentage of an organic component, such that after sample loading, the sorbent can be washed thoroughly with an aqueous-organic binary solvent containing a reasonably high percentage of organic. Such a wash would furnish a clean extract by removing unwanted matrix components, which can interfere with mass spectrometric detection and cause ion suppression. An SPE protocol employing this sorbent would preferably comprise a simple procedure for elution of the desired analyte, such that the eluting solvent is compatible with mass spectrometric mode of detection and if necessary, be adapted to be injected directly into an LC/MS/MS system. Further, such a sorbent would preferably be easily solvated with an aqueous solvent (e.g. water or buffer), remain solvated for a long period of time and would display comparable SPE behavior under wet or dry conditions. Such an SPE procedure/format would preferably be compatible with the high throughput screening of large volume of samples commonly employed in the pharmaceutical industry. These and other problems are solved by the compositions and methods of present invention.